Biomarker Detection Methods and Systems and Kits for Practicing Same

ABSTRACT

Aspects of the present disclosure include methods that include co-culturing a cell and a microparticle that includes a capture ligand, in a culture medium under conditions in which a biomarker produced by the cell is bound by the capture ligand. Such methods may further include detecting (e.g., by flow or mass cytometry) complexes that include the microparticle, the capture ligand, the biomarker, and a detection reagent. The methods may further include determining the proportion or number of cells among a heterogeneous cell population that produced the biomarker and/or the level of biomarker secreted by such cells. Compositions, systems and kits are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/336,439, filed Oct. 27, 2016, which claims priority to the filingdate of the U.S. Provisional Patent Application Ser. No. 62/253,549,filed Nov. 10, 2015, the disclosure of which is herein incorporated byreference.

INTRODUCTION

The measurement of soluble and secreted cytokines and other analytes inserum and plasma is becoming increasingly important in the study andmanagement of many diseases. Immune cells can be stimulated by variousmeans to produce specific analytes. Secreted analytes can bequantitatively measured by ELISA or Cytometric Bead Array (CBA). Thefrequencies of cytokine-producing cells can be measured by Enzyme-LinkedImmunospot (ELISPOT) or by flow cytometric analysis of intracellularcytokines.

The ELISPOT assay is a widely used method for monitoring cellular immuneresponses in humans and other animals, and has found clinicalapplications in the diagnosis of tuberculosis and the monitoring ofgraft tolerance or rejection in transplant patients. The ELISPOTtechnique has proven to be among the most useful means available formonitoring cell-mediated immunity, due to its ability to detect rareantigen-specific T cells (or B cells) and its ability to visualizesingle positive cells within a population of peripheral bloodmononuclear cells (PBMCs). The ELISPOT assay allows visualization ofindividual activated or responding cells and identification of thesecretory product(s) released. Each spot that develops in the assaytheoretically represents a single reactive cell, although multiple cellson top of one another cannot be distinguished. Thus, the ELISPOT assayprovides both qualitative (regarding the specific cytokine or othersecreted immune molecule) and quantitative (the frequency of respondingcells within the test population) information. ELISA-based assays havelong been the standard for quantitative analysis of cytokines and otherbiomarkers, but are not well suited for high throughput multiplexanalyses and suffer from poor sensitivity due to dilution by diffusionin the supernatant.

The introduction of flow cytometric bead-based technology has added anew approach for investigators to simultaneously measure multipleanalytes in biological and environmental samples. In the context ofmeasuring secreted factors, existing cytometric bead-based assaysinvolve culturing cells that secrete the factor(s) of interest,separating the supernatant containing the factor(s) from the cells,combining the supernatant with beads adapted to bind to the factor(s)(e.g., via an antibody linked to the beads), and detecting the resultingcomplexes in a flow cytometer.

The Cytometric Bead Array (CBA) system from BD Biosciences (San Jose,Calif.) relies on different fluorescent intensities of a singlefluorophore to accomplish multiplexing. The xMAP® technology (formerlyLabMAP, FlowMetrix) by Luminex (Austin, Tex.) uses digital signalprocessing capable of classifying polystyrene beads (microspheres) dyedwith distinct proportions of red and near-infrared fluorophores. Theseproportions define ‘spectral addresses’ for each bead population.Copalis® multiplex technology, produced by DiaSorin, is unique from mostother multiplex bead array approaches in that it does not usefluorescence to discriminate different bead populations, but ratherdifferentiates monomeric latex microspheres from latex aggregates andcells on the basis of their unique light scatter properties by flowcytometry. The system can measure two types of events:polystyrene-microparticle latex co-agglutination and polystyrene-goldcolloid microparticle coupling. The former is useful for detecting thepresence of antibodies to infectious agents or autoantigens, which arecoated onto latex microparticles. Further details regarding bead-basedassays may be found, e.g., in Elshal & McCoy (2006) Methods38(4):317-323.

SUMMARY

Aspects of the present disclosure include methods that includeco-culturing a cell and a microparticle that includes a capture ligand,in a culture medium under conditions in which a biomarker produced bythe cell is bound (e.g., immediately bound) by the capture ligand. Suchmethods may further include detecting (e.g., by flow or mass cytometry)complexes that include the microparticle, the capture ligand, thebiomarker, and a detection reagent. The methods may further includedetermining the proportion or number of cells among a heterogeneous cellpopulation that produced the biomarker and/or the level of biomarkersecreted by such cells. Compositions, systems and kits are alsoprovided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, panels A, B and C, schematically illustrate methods according toembodiments of the present disclosure.

FIG. 2 schematically illustrates a method for detecting antigen-specificcellular responses according to one embodiment of the presentdisclosure.

FIG. 3 shows the detection of phytohaemagglutinin (PHA)-inducedinterferon gamma (IFNγ) spots by ELISPOT and a FlowSpot assay accordingto one embodiment of the present disclosure.

FIG. 4 shows the number of spots counted at various concentrations ofPHA by ELISPOT and a FlowSpot assay according to one embodiment of thepresent disclosure.

FIG. 5 shows flow cytometric data relating to viral peptide-specific Tcell detection according to one embodiment of the present disclosure.

FIG. 6 provides flow cytometric data relating to two-color detection ofIFNγ and interleukin-2 (IL-2) according to one embodiment of the presentdisclosure.

FIG. 7, Panels A and B, provides flow cytometric data relating tothree-color detection of IFN-γ, IL-2 and Granzyme B (GrB) according toone embodiment of the present disclosure.

FIG. 8 schematically illustrates a method according to one embodiment ofthe present disclosure for antigen-specific memory B cell detection.

FIG. 9 shows data relating to HLA antigen specific memory B celldetection using the method illustrated in FIG. 8.

FIG. 10 provides data showing the effects of immunotherapeutic agents onthe secretion of the Th1 cytokine, IFN-γ, as determined using a methodaccording to one embodiment of the present disclosure.

FIG. 11 provides data showing the effects of immunotherapeutic agents onthe secretion of the Th1 cytokine, IL-2, as determined using a methodaccording to one embodiment of the present disclosure.

FIG. 12 provides data showing the effects of immunotherapeutic agents onthe secretion of the Th2 cytokine, IL-4, as determined using a methodaccording to one embodiment of the present disclosure.

FIG. 13 provides data showing the effects of immunotherapeutic agents onthe secretion of the Th2 cytokine, IL-5, as determined using a methodaccording to one embodiment of the present disclosure.

FIG. 14 provides data showing the effects of immunotherapeutic agents onthe secretion of the Th2 cytokine, IL-10, as determined using a methodaccording to one embodiment of the present disclosure.

FIG. 15 shows data in which antigen-specific immunity to viral andbacterial peptides and proteins was assessed (based on IFN-γ secretion)using a method according to one embodiment of the present disclosure.

FIG. 16 shows data in which antigen-specific immunity to viral andbacterial peptides and proteins was assessed (based on IL-2 secretion)using a method according to one embodiment of the present disclosure.

FIG. 17 shows data in which antigen-specific immunity to viral andbacterial peptides and proteins was assessed (based on IL-4 secretion)using a method according to one embodiment of the present disclosure.

FIG. 18 shows data in which antigen-specific immunity to viral andbacterial peptides and proteins was assessed (based on IL-5 secretion)using a method according to one embodiment of the present disclosure.

FIG. 19 shows data in which antigen-specific immunity to viral andbacterial peptides and proteins was assessed (based on IL-10 secretion)using a method according to one embodiment of the present disclosure.

FIG. 20 shows data from seven individuals in which native viralpeptide/antigen-specific T cell responses were assessed (based on IFN-γand IL-2 secretion) using a method according to one embodiment of thepresent disclosure.

FIG. 21 shows data from seven individuals in which viruspeptide/antigen-specific T cell responses were assessed (based on IL-4and IL-5 secretion) using a method according to one embodiment of thepresent disclosure.

FIG. 22 shows data from seven individuals in which viruspeptide/antigen-specific T cell responses were assessed (based on IL-10secretion) using a method according to one embodiment of the presentdisclosure.

FIG. 23 illustrates the principle of a multiplex HLA antigen-specificmemory B cell assay according to one embodiment of the presentdisclosure.

FIG. 24 illustrates the principle of a multiplex HLA antigen-specificmemory B cell assay according to one embodiment of the presentdisclosure.

FIG. 25 shows the results of a multiplex HLA antigen-specific memory Bcell assay as illustrated in FIGS. 23 and 24.

FIG. 26 shows the results of a multiplex HLA antigen-specific memory Bcell assay as illustrated in FIGS. 23 and 24.

FIG. 27 shows the results of a multiplex HLA antigen-specific memory Bcell assay as illustrated in FIGS. 23 and 24.

FIG. 28 shows the results of a multiplex HLA antigen-specific memory Bcell assay as illustrated in FIGS. 23 and 24.

FIG. 29 provides flow cytometry data comparing the results of acytometric bead array (CBA) assay to the results produced using aFlowSpot assay according to one embodiment of the present disclosure.

FIG. 30 provides flow cytometry data relating to the determination ofrecipient immunity to donor cells using a FlowSpot assay according toone embodiment of the present disclosure.

DETAILED DESCRIPTION

Provided are methods that include co-culturing a cell and amicroparticle that includes a capture ligand, in a culture medium underconditions in which a biomarker produced by the cell is bound by thecapture ligand. Such methods may further include detecting (e.g., byflow or mass cytometry) complexes that include the microparticle, thecapture ligand, the biomarker, and a detection reagent. The methods mayfurther include determining the proportion or number of cells among apurified or heterogeneous cell population that produced the biomarkerand/or the level of biomarker secreted by such cells. Compositions,systems and kits are also provided.

Before the methods, compositions, systems and kits of the presentdisclosure are described in greater detail, it is to be understood thatthe methods, compositions, systems and kits are not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the methods, compositions, systemsand kits will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the methods, compositions, systemsand kits. The upper and lower limits of these smaller ranges mayindependently be included in the smaller ranges and are also encompassedwithin the methods, compositions, systems and kits, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the methods, compositions,systems and kits.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods, compositions, systems and kits belong.Although any methods, compositions, systems and kits similar orequivalent to those described herein can also be used in the practice ortesting of the methods, compositions, systems and kits, representativeillustrative methods, compositions, systems and kits are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the materials and/or methods in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present methods, compositions, systems and kits arenot entitled to antedate such publication, as the date of publicationprovided may be different from the actual publication date which mayneed to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the methods, compositions,systems and kits, which are, for clarity, described in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features of the methods, compositions,systems and kits, which are, for brevity, described in the context of asingle embodiment, may also be provided separately or in any suitablesub-combination. All combinations of the embodiments are specificallyembraced by the present disclosure and are disclosed herein just as ifeach and every combination was individually and explicitly disclosed, tothe extent that such combinations embrace operable processes and/orcompositions/kits. In addition, all sub-combinations listed in theembodiments describing such variables are also specifically embraced bythe present methods, compositions, systems and kits and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein. As will be apparent to those of skillin the art upon reading this disclosure, each of the individualembodiments described and illustrated herein has discrete components andfeatures which may be readily separated from or combined with thefeatures of any of the other several embodiments without departing fromthe scope or spirit of the present methods, compositions, systems andkits. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Methods

Aspects of the present disclosure include methods that includeco-culturing a cell and a microparticle that includes a capture ligand,in a culture medium under conditions in which a biomarker produced bythe cell is bound by the capture ligand on the microparticle.

According to certain embodiments, the methods of the present disclosureinclude co-culturing in a culture medium: a heterogeneous cellpopulation that includes a first subpopulation of cells and a secondsubpopulation of cells, and microparticles that include capture ligandsthat specifically bind to a biomarker of interest. Such methods mayfurther include stimulating the first subpopulation of cells to secretethe biomarker, where upon secretion of the biomarker, the biomarker isbound by the capture ligands.

Embodiments of the above methods will now be described in detail. Assummarized above, the cells and microparticles are co-cultured in aculture medium under conditions in which a biomarker produced by thecell is bound by the capture ligand. By “co-culturing” is meant that thecells are cultured (e.g., grown/maintained under controlled conditions)in the presence of the microparticles.

The co-culture may be in suspension, where the cells and microparticlesare not attached to a surface of a cell culture container during theco-culture. In other aspects, the co-culture may be an adherentco-culture, in which the cells are attached to a surface of a cellculture container. In this way, the cells and microparticles arespatially “fixed” relative to one another during the co-culture, withsome microparticles being in the immediate vicinity of the cells andother microparticles being remote from the cells (and hence lessaccessible (or completely inaccessible) to any biomarkers secreted bythe cells). In certain aspects, the cells are mixed with microparticlesand seeded on the bottom of a culture container by centrifugation and/orfree sedimentation.

Suitable conditions for culturing one or more cell types of interest aredescribed in detail, e.g., in Aschner et al. Cell Culture Techniques(ISBN 978-1-61779-077-5); Mitry & Hughes Human Cell Culture Protocols(ISBN 978-1-61779-367-7); Freshney, R. I. Culture of Animal Cells (ISBN:978-O-470-52812-9); Mather Stem Cell Culture (ISBN-13: 9780080878041);Coleman et al. Plant Cell Culture (ISBN: 9781859963203); and elsewhere.Optimal conditions may vary depending on the particular cell type(s)being cultured. Example conditions for certain cell types are describedin the Experimental section below.

The co-culture conditions include co-culturing the cells andmicroparticles at a suitable temperature (such as from 30° C. to 45° C.,e.g., 37° C.), and gas mixture (e.g., from 3% to 10% CO₂, e.g., 5% CO₂).The co-culture may occur in a suitable culture medium for the cell typebeing cultured. Culture media that find use in culturing cells are knownand include, e.g., DMEM, RPMI, and the like.

The culture medium may be selected or modified to have a suitable pH.Most normal mammalian cell lines grow well at pH 7.4, and there is verylittle variability among different cell strains. However, sometransformed cell lines have been shown to grow better at slightly moreacidic environments (e.g., pH 7.0-7.4), and some normal fibroblast celllines prefer slightly more basic environments (e.g., pH 7.4-7.7). Insectcell lines such as Sf9 and Sf21 grow optimally at pH 6.2.

The culture medium may be selected or modified to include additionalcomponents, such as glucose, growth factors, other nutrients,antibiotics, and/or any other components useful for growing/maintainingthe cell type(s) of interest. The growth factors used to supplementmedia may be derived from the serum of animal blood, such as fetalbovine serum (FBS), bovine calf serum, equine serum, and porcine serum.Alternatively, serum-free medium may be used. Antibiotics useful forcontrolling or eliminating cell culture contamination include, but arenot limited to, actinomycin D, kanamycin, ampicillin, neomycin,carbenicillin, penicillin streptomycin (Pen Strep), cefotaxime,polymyxin B, fosmidomycin, streptomycin, and gentamicin.

The co-culture occurs under conditions in which the biomarker producedby the cell is bound by the capture ligand. The co-culture conditions,therefore, include a temperature, pH, etc. such that the conditions arecompatible with the binding of the capture ligand to the biomarker,which conditions may vary depending on the particular biomarker and thecapture ligand employed when practicing the methods of the presentdisclosure. Such conditions may be the conditions under which the celltype(s) of interest would otherwise be cultured for standardgrowth/maintenance.

The ratio of cells to microparticles in the co-culture may vary. Incertain aspects, the number of cells is greater than the number ofmicroparticles, and the cell:microparticle ratio at the onset of theco-culture is from 1:1 to 1000:1, including 2:1 to 50:1, such as from5:1 to 30:1, including 8:1 to 20:1, e.g., 10:1 to 17:1 (e.g., 14:1).According to certain embodiments, when the number of cells is greaterthan the number of microparticles, the ratio of cells to microparticlesat the onset of the co-culture is 2:1 or greater, 3:1 or greater, 4:1 orgreater, 5:1 or greater, 6:1 or greater, 7:1 or greater, 8:1 or greater,9:1 or greater, 10:1 or greater, 11:1 or greater, 12:1 or greater, 13:1or greater, 14:1 or greater, 15:1 or greater, 16:1 or greater, 17:1 orgreater, 18:1 or greater, 19:1 or greater, or 20:1 or greater. Incertain aspects, when the number of cells is greater than the number ofmicroparticles, the ratio of cells to microparticles at the onset of theco-culture is 20:1 or less, 19:1 or less, 18:1 or less, 17:1 or less,16:1 or less, 15:1 or less, 14:1 or less, 13:1 or less, 12:1 or less,11:1 or less, 10:1 or less, 9:1 or less, 8:1 or less, 7:1 or less, 6:1or less, 5:1 or less, 4:1 or less, 3:1 or less, or 2:1 or less.

According to certain embodiments, the number of microparticles isgreater than the number of cells in the co-culture. For example, theratio of microparticles to cells at the onset of the co-culture may befrom 2:1 to 2000:1, including 2:1 to 200:1, such as from 5:1 to 50:1,including 8:1 to 20:1, e.g., 10:1. In certain aspects, when the numberof microparticles is greater than the number of cells, the ratio ofmicroparticles to cells at the onset of the co-culture is 2:1 orgreater, 3:1 or greater, 4:1 or greater, 5:1 or greater, 6:1 or greater,7:1 or greater, 8:1 or greater, 9:1 or greater, 10:1 or greater, 11:1 orgreater, 12:1 or greater, 13:1 or greater, 14:1 or greater, 15:1 orgreater, 16:1 or greater, 17:1 or greater, 18:1 or greater, 19:1 orgreater, 20:1 or greater, 50:1 or greater, or 200:1 or greater.According to certain embodiments, when the number of microparticles isgreater than the number of cells, the ratio of microparticles to cellsat the onset of the co-culture is 200:1 or less, 50:1 or less, 20:1 orless, 19:1 or less, 18:1 or less, 17:1 or less, 16:1 or less, 15:1 orless, 14:1 or less, 13:1 or less, 12:1 or less, 11:1 or less, 10:1 orless, 9:1 or less, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less,4:1 or less, 3:1 or less, or 2:1 or less.

The cells to be co-cultured with the microparticles may be derived froma source of interest, such as a biological sample. In certain aspects,the cells are obtained from a biological fluid or biological tissue.Examples of biological fluids include blood, plasma, serum, saliva,urine, semen, stool, sputum, cerebral spinal fluid, tears, mucus,amniotic fluid or the like. Biological tissues are aggregates of cells,usually of a particular kind together with their intercellular substancethat form one of the structural materials of a human, animal, plant,bacterial, fungal or viral structure, including connective, epithelium,muscle and nerve tissues. Examples of biological tissues also includeorgans, tumors, lymph nodes, arteries, buccal tissue (e.g., the cellsmay be obtained from a buccal swab), biopsy tissue, and individualcells.

In certain aspects, the cells to be co-cultured are obtained from afluid, tissue, organ, and/or the like of a mammal (e.g., a human, arodent (e.g., a mouse), or any other mammal of interest). In otheraspects, the cells to be co-cultured are obtained from a source otherthan a mammal, such as bacteria, yeast, insects (e.g., drosophila),amphibians (e.g., frogs (e.g., Xenopus)), plants, or any othernon-mammalian cellular source. According to certain embodiments, viruses(rather than cells) are co-cultured with the microparticles.

The cells may be obtained from an individual (e.g., a human individual)of interest. According to certain embodiments, the individual is one inwhich it is desirable to predict and monitor vaccine efficacy before orafter vaccination. As such, encompassed by the present disclosure aremethods of predicting and monitoring vaccine efficacy before or aftervaccination. In certain aspects, the individual is one in which it isdesirable to evaluate a given specific immunity to a pathogenic organismincluding bacteria, viruses, fungi, and/or parasites for the purposes oftreatment or diagnosing disease susceptibility. Accordingly, encompassedby the present disclosure are methods of determining a given specificimmunity of an individual to a pathogenic organism including bacteria,viruses, fungi, and/or parasites, and optionally diagnosing and/ortreating the individual accordingly. In certain aspects, the individualis one in which it is desirable to evaluate a given specific immunity toan allergen (e.g., peanuts, ragweed pollen, etc.) for purposes oftreatment or diagnosing allergy susceptibility. As such, encompassed bythe present disclosure are methods of determining a given specificimmunity of an individual to an allergen, and optionally diagnosingand/or treating the individual accordingly. According to certainembodiments, the individual is an immunotherapy patient (e.g., a cancerpatient, an auto-immune disease patient, etc.) where it is desirable toevaluate and monitor the potential/actual efficacy of immunotherapy(immunotherapeutic drugs) for the purposes of treatment or diseaseclassification. As such, encompassed by the present disclosure aremethods of determining/monitoring in an immunotherapy patientpotential/actual efficacy of immunotherapy, and optionally diagnosingand/or treating the patient accordingly. In certain aspects, theindividual is a transplant candidate or transplant recipient in which itis desirable to evaluate the given specific immunity or thecompatibility between the potential/actual graft donor and recipient,assess risk for transplant rejection with a given donor, monitor graftrejection, and/or guide immunotherapy. Encompassed by the presentdisclosure, therefore, are methods of assessing risk for transplantrejection with a given donor, monitoring graft rejection, and/or guidingimmunotherapy. According to certain embodiments, the individual is atumor patient in which it is desirable to detect membrane-bound orsecreted tumor markers for purposes of treatment or diseaseclassification. As such, encompassed by the present disclosure aremethods of treating a tumor patient and/or classifying a tumor of apatient, where the method includes detecting membrane-bound or secretedtumor markers according to the methods of the present disclosure. Incertain aspects, the individual is a tumor patient in which it isdesirable to monitor tumor metastasis by detecting circulating tumorcells (CTC) for the purposes of treatment or disease classification.Accordingly, aspects of the present disclosure include methods oftreating a tumor patient, monitoring tumor metastasis, and/orclassifying a tumor of a patient, where the method includes detectingcirculating tumor cells (CTC) in the patient using the methods of thepresent disclosure.

In certain aspects, the cell co-cultured with the microparticles isselected from a peripheral blood mononuclear cell (PBMC), a white cell,a tumor cell, a stem cell, an immune cell, a lymphocyte, a T cell, a Bcell, a natural killer (NK) cell, a natural killer T (NKT) cell, amacrophage, a dendritic cell, a monocyte, a granulocyte, an epithelialcell, an endothelial cell, and a platelet.

According to certain embodiments, the cells to be co-cultured are immunecells, such as lymphocytes (e.g., T cells, B cells (e.g., memory Bcells), plasma cells, natural killer (NK) cells, and/or natural killer T(NKT) cells), macrophages, dendritic cells, monocytes, platelets, or anycombination thereof.

T cells have a variety of roles and are classified by subsets. T cellsare divided into two broad categories: CD8+ T cells or CD4+ T cells,based on which protein is present on the cell's surface. T cells carryout multiple functions, including killing infected cells and activatingor recruiting other immune cells. CD8+ T cells also are called cytotoxicT cells or cytotoxic lymphocytes (CTLs). CTLs have specializedcompartments, or granules, containing cytotoxins that cause apoptosis,i.e., programmed cell death. Because of its potency, the release ofgranules is tightly regulated by the immune system. The four major CD4+T-cell subsets are TH1, TH2, TH17, and Treg, with “TH” referring to “Thelper cell.” TH1 cells interact with professional antigen presentingcells (the cells whose function it is to present antigen) and coordinateimmune responses against pathogens. They produce and secrete moleculesthat alert, recruit, and activate other immune cells to respond (e.g.,CD8+ T cells/CTL). TH2 cells interact with B cells and direct the immuneresponse against extracellular pathogens toward antibody secretion. TH17cells are important for recruiting neutrophils and are named for theirability to produce interleukin 17 (IL-17), a signaling molecule thatactivates immune and non-immune cells.

B cells function to present antigens to T cells, and to produceantibodies to neutralize infectious microbes, other foreign substances,cryptic antigens (e.g., DNA), or other antibodies. Antibodies areexpressed in two ways. The B-cell receptor (BCR), which is present onthe surface of a B cell, is a membrane bound antibody. B cells (anddifferentiated plasma cells) also secrete antibodies to diffuse and bindto pathogens and other foreign antigens. This dual expression isimportant because the initial problem, e.g., a bacterium, is recognizedby a unique BCR and activates the B cell. The activated B cell respondsby secreting antibodies, essentially the BCR but in soluble form.

In certain aspects, the cells to be co-cultured are obtained from atumor. “Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer” and “cancerous” refer toor describe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth/proliferation. Examples ofcancer include but are not limited to, carcinoma, lymphoma, myeloma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung, squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma, various types of head andneck cancer, and the like. In certain aspects, the cells to beco-cultured are circulating tumor cells (CTCs). In certain aspects, thecells to be co-cultured are obtained from a pre-cancerous tissue, suchas the relevant tissue of an individual having a pre-cancerouscondition, such as myelodysplastic syndrome (MDS) or the like.

As summarized above, the cells of interest are co-cultured withmicroparticles. By “microparticle” is meant a small particle (which isnot a cell), having a greatest dimension ranging from 0.001 μm to 1000μm, such as from 0.5 μm to 100 μm, e.g., 0.1 μm to 20 μm. In certainaspects, the microparticle has a greatest dimension of 20 μm or less,such as 15 μm or less, 10 μm or less, 5 μm or less, 1 μm or less, 0.75μm or less, 0.5 μm or less, 0.4 μm or less, 0.3 μm or less, 0.2 μm orless, 0.1 μm or less, 0.01 μm or less, or 0.001 μm or less.

The microparticles may have any suitable shape, including but notlimited to spherical, spheroid, rod-shaped, disk-shaped, pyramid-shaped,cube-shaped, cylinder-shaped, nanohelical-shaped, nanospring-shaped,nanoring-shaped, arrow-shaped, teardrop-shaped, tetrapod-shaped,prism-shaped, or any other suitable geometric or non-geometric shape.

The microparticles may be made of any suitable material, including butnot limited to, latex, polystyrene, silica, a magnetic material, aparamagnetic material, or any combination thereof.

The microparticles include capture ligands present on (e.g., covalentlyattached to) the surface thereof. By “capture ligand” is meant a bindingpartner (e.g., a specific binding partner) for the biomarker, in whichthe capture ligand binds (or “captures”) the biomarker upon contactingthe biomarker under the co-culture conditions. Capture ligands that finduse in practicing the methods of the present invention include, but arenot limited to, an antibody (e.g., when the biomarker is an antigen orantibody to which the antibody specifically binds), an antigen (e.g.,when the biomarker is an antibody that specifically binds the antigen),an enzyme (e.g., when the biomarker is a specific substrate of theenzyme), a substrate (e.g., when the biomarker is an enzyme thatspecifically acts upon the substrate), a protein, a peptide, a nucleicacid (e.g., when the biomarker is complementary nucleic acid), a drug, achemical compound, and any combination thereof. For example, when themethod involves detecting the secretion of a cytokine from the cells ofinterest, the capture ligand on the surface of the microparticles may bean antibody that specifically binds the cytokine. Also by way ofexample, when the method involves detecting the presence of proteins onthe surface of a cell (e.g., B cell receptors (BCRs) on the surface ofmemory B cells) or membrane bound cytokines on the surface of a cell,the capture ligand on the surface of the microparticles may be a ligandto which the protein on the surface of the cell binds. For example, whenthe cells co-cultured with the microparticles are memory B cells, thecapture ligand on the surface of the microparticles may be an antigen(e.g., an HLA antigen) to which the BCR specifically binds. In certainaspects, the capture ligand is a cluster of differentiation (CD)molecule, a tumor marker, a carbohydrate, a lipid, a peptide, a vitamin,a small chemical/molecule, or a binding partner (e.g., an antibody) thatbinds (e.g., specifically binds) to a cluster of differentiation (CD)molecule, a tumor marker, a carbohydrate, a lipid, a peptide, a vitamin,a small chemical/molecule, or the like.

The terms “specific binding,” “specifically binds,” and the like, referto the preferential binding to a molecule relative to other molecules ormoieties in a solution or reaction mixture. In some embodiments, theaffinity between binding member and the target analyte to which itspecifically binds when they are specifically bound to each other in abinding complex is characterized by a K_(d) (dissociation constant) of10⁻⁶ M or less, such as 10⁻⁷ M or less, including 10⁻⁸ M or less, e.g.,10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹³M or less, 10⁻¹⁴ M or less, including 10⁻¹⁵ M or less.

“Affinity” refers to the strength of binding, increased binding affinitybeing correlated with a lower K_(d). As such, “binds specifically” or“specifically binds” is not meant to preclude a given binding memberfrom binding to more than one analyte of interest. For example,antibodies that bind specifically to an analyte polypeptide of interestmay be capable of binding other polypeptides at a weak, yet detectable,level (e.g., 10% or less of the binding shown to the polypeptide ofinterest). Such weak binding, or background binding, is readilydiscernible from the specific antibody binding to the polypeptide ofinterest, e.g., by use of appropriate controls.

As used herein, the term “antibodies” includes antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab,F(ab′)₂, Fv, scFv, bi-specific-scFv, diabody, Fd, and Fc fragments,chimeric antibodies, humanized antibodies, fully human antibodies,single-chain antibodies, and fusion proteins including anantigen-binding portion of an antibody and a non-antibody protein.

The biomarker may be any biomarker of interest. For example, thebiomarker may be a biomarker present within and secreted by the cells.Alternatively, the biomarker may be a biomarker expressed and/or presenton (e.g., attached to) the surface of the cell. Biomarkers present onthe surface of cells include, but are not limited to, cell surfacereceptors, such as an immunoglobulin in the case of B cell receptorspresent on the surface of memory B cells. In certain aspects, thebiomarker captured by the capture ligand is a cytokine, animmunoglobulin (e.g., an antibody), a hormone, a growth factor, anenzyme, a protease, a protein, a heat shock protein, a glycoprotein, apeptide, a nucleic acid, a drug, a cluster differentiation (CD)molecule, a tumor marker, a receptor, a phosphorylated cell signalingprotein, a complement component, a perforin, a nucleic acid, or anycombination thereof. When the biomarker is a cytokine, the cytokine maybe an interferon (e.g., IFN-γ, etc.), a chemokine, an interleukin (e.g.,IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17 etc.), a lymphokine, a tumornecrosis factor (e.g., TNF-α, etc.), transforming growth factor β(TGFβ), and the like, or any combination thereof. In certain aspects,the biomarker is a response element, such as perforin, granzyme B, orthe like.

When the biomarker is secreted from the cells, the methods may includestimulating the cells to secrete the biomarker. In certain aspects,stimulating the cells to secrete the biomarker includes adding astimulant to the culture medium. Various stimulants for stimulating thesecretion of particular biomarkers from particular cell types ofinterest are known. In some embodiments, the stimulant is an antigen, aligand, a protein, a lectin, a nucleic acid, a sub-cellular component, amicroorganism (e.g., a bacteria, a virus, etc.) or component thereof(e.g., a bacterial or viral peptide), a chemical compound, an agonist,an antagonist, an enzyme, a drug, a vaccine, a CD (clusterdifferentiation) molecule, a receptor, and combinations thereof. Thestimulant is provided in the culture medium at a concentrationsufficient to stimulate the cells to secrete the biomarker. Specificexamples of stimulants and conditions suitable for eliciting thesecretion of biomarkers of interest from cells of interest are describedin the Experimental section below.

According to certain embodiments, when the methods include stimulatingthe cell to secrete the biomarker, the cell and the microparticle areco-cultured in the presence of cells that are not stimulated to secretethe biomarker. For example, the cells in the co-culture may constitute aheterogeneous cell population that includes a first subpopulation ofcells and a second subpopulation of cells, where only the firstsubpopulation of cells is of a type that secretes the biomarker uponbeing contacted by the stimulant.

In certain aspects, the methods include lysing the cells upon binding ofthe biomarker to the capture ligands. Any suitable approach for lysingthe cells in the co-culture may be employed. According to certainembodiments, lysing the cells includes the addition of a lysis buffer(e.g., NP-40 lysis buffer) to the co-culture after the biomarker isbound to the capture ligands. Such a lysis step may be employed tofacilitate certain downstream applications, such as detection ofcomplexes that include the microparticle, capture ligand, biomarker anda detection reagent in a flow or mass cytometer.

According to certain embodiments, the methods include, after thebiomarker is bound by the capture ligand, contacting the biomarker witha detection reagent to form a complex including the microparticle, thecapture ligand, the biomarker, and the detection reagent. The detectionreagent may be a binding partner (e.g., a specific binding partner) forthe biomarker, which detection reagent is capable of binding to thebiomarker when the biomarker is also bound by the capture ligand.According to certain embodiments, the detection reagent is an antibody(e.g., an antibody that specifically binds the biomarker), an antigen, aligand, a protein, a receptor, a peptide, an enzyme, a substrate, anucleic acid, a drug, a chemical compound, a carbohydrate, andcombinations thereof.

In certain aspects, the detection reagent includes a detectable label.Detectable labels that find use in practicing the subject methodsinclude, but are not limited to, fluorescent labels, metal elements(e.g., for use in a mass cytometry instrument), radiolabels, luminescentagents, and the like. For example, when it is desirable to detect thecomplex in a flow cytometer, a fluorescent label suitable/compatiblewith the particular flow cytometer may be employed. Detection reagents(e.g., antibodies, antigens, etc.) having such labels are available andinclude, but are not limited to, phycoerythrin (PE or R-PE), FITC,Cy-Chrome™ dye (BD Biosciences, San Jose, Calif.), peridinin chlorophyllprotein (PerCP), allophycocyanin (APC), APC-Cy7, Alexa Fluor 488, AlexaFluor 633, and the like.

Methods (generally referred to herein as “FlowSpot” assays) according toembodiments of the present disclosure are schematically illustrated inFIG. 1, panels A, B and C. Shown in panel A is a cell co-cultured withmicroparticles. In this example, a stimulant is added to the co-culturemedium, which stimulant stimulates (e.g., activates) the cell to secretea biomarker. The microparticles include a capture ligand thatspecifically binds to the biomarker. The microparticles adjacent (or inclose proximity) to the responding stimulated cells are exposed to ahigher concentration of the secreted biomarker, and therefore willcapture more biomarker than microparticles more distant to thestimulated cells or to microparticles adjacent to non-responsive cells.In this example, the cell is lysed after stimulation. Following celllysis, complexes that include the microparticle, capture ligand, andbiomarker are contacted with a labeled detection reagent (e.g., afluorescently-labeled antibody) that specifically binds the biomarker(e.g., if the capture ligand itself is not already labeled), formingcomplexes that include the microparticle, the capture ligand, thebiomarker, and the labeled detection reagent.

The microparticles are harvested and acquired on a flow or masscytometer. The number of positive complexes (“events” or “spots”)detected during flow or mass cytometric analysis may be counted.

Shown in panel B is a heterogeneous cell population that includes afirst subpopulation of cells and a second subpopulation of cells,co-cultured with microparticles. A stimulant is added to the co-culturemedium, which stimulant only stimulates (e.g., activates) cells of thefirst subpopulation of cells to secrete a biomarker. The microparticlesinclude a capture ligand that specifically binds to the biomarker. Themicroparticles adjacent (or in close proximity) to the respondingstimulated cells are exposed to a higher concentration of the secretedbiomarker, and therefore will capture more biomarker than microparticlesmore distant to the stimulated cells or to microparticles adjacent tonon-responsive cells. In this example, the cells are lysed afterstimulation. Following cell lysis, complexes that include themicroparticle, capture ligand, and biomarker are contacted with alabeled detection reagent (e.g., a fluorescently-labeled antibody) thatspecifically binds the biomarker (e.g., if the capture ligand itself isnot already labeled), forming complexes that include the microparticle,the capture ligand, the biomarker, and the labeled detection reagent.The microparticles are harvested and acquired on a flow or masscytometer. The number of positive microparticles (“events” or “spots”)detected during flow or mass cytometric analysis may be counted, and thenumber of microparticles counted may be used to determine the number ofcells that were stimulated in the co-culture and/or the proportion(e.g., percentage) of responding cells within the heterogeneous cellpopulation. The signal intensity (e.g., Median Fluorescence Intensity;MFI) of positive microparticles may be used to determine concentrationof the biomarker secreted from responding cells; or biomarker secretingcapability of responding cells. Microparticles that were adjacent (or inclose proximity) to the stimulated cells during the co-culture will havea higher detectable signal by virtue of the greater “load” ofbiomarker/labeled detection reagent on the surface thereof, as comparedto microparticles that were more distant to the stimulated cells in theco-culture.

Shown in panel C is a heterogeneous cell population that includes afirst donor cell and a second donor cell, co-cultured withmicroparticles. In this example, the first donor cell activates thesecond donor cell, which activation causes the second donor cell tosecrete a biomarker. The microparticles include a capture ligand thatspecifically binds to the biomarker. The microparticles adjacent (or inclose proximity) to the activated donor cell are exposed to a higherconcentration of the secreted biomarker, and therefore will capture morebiomarker than microparticles more distant to the activated cell or tomicroparticles adjacent to any non-activated cells in the co-culture. Inthis example, the cells are lysed after the co-culture. Following celllysis, complexes that include the microparticle, capture ligand, andbiomarker are contacted with a labeled detection reagent (e.g., afluorescently-labeled antibody) that specifically binds the biomarker(e.g., if the capture ligand itself is not already labeled), formingcomplexes that include the microparticle, the capture ligand, thebiomarker, and the labeled detection reagent. The microparticles areharvested and acquired on a flow or mass cytometer. The number ofpositive complexes (“events” or “spots”) detected during flow or masscytometric analysis may be counted.

In any embodiment in which flow or mass cytometric analysis is employed,the cytometric analysis may include—in addition to counting the numberof positive complexes (“events” or “spots”) detected—determining asignal intensity for each detected complex. Such signal intensity datamay be used to determine the mean or median signal intensity of thedetected complexes, which mean or median signal intensity may be used todetermine a level of biomarker secretion (and, therefore, level of cellstimulation/activation) in the co-culture. Such information is notprovided by existing bead-based approaches for detecting cellstimulation/activation, such as the existing cytometric bead array (CBA)approach.

A detection scheme according to one embodiment of the present disclosureis schematically illustrated in FIG. 2. In this example,antigen-specific cell responses are detected. A population of cells isco-cultured with microparticles, and cells (e.g., a subpopulation ofcells among a heterogeneous cell population) are stimulated by additionof an antigen to the culture medium. The stimulated cells secrete abiomarker (e.g., a cytokine), which biomarker is captured by captureligands present on the surface of the microparticles. The biomarker isthen contacted with a labeled detection reagent (in this example, afluorescent reporter), and the resulting complexes are acquired on aflow cytometer and detected, e.g., to determine the number and/orproportion of cells activated by the antigen in the co-culture based onthe number of positive spots, and/or the level of activation based onthe fluorescence intensity of the positive spots.

A further example FlowSpot method according to one embodiment of thepresent disclosure is schematically illustrated in FIG. 8. In thisexample, a sample containing memory B cells is co-cultured withmicroparticles and interrogated to determine whether the sample includesmemory B cells having B cell receptors (BCRs) that specifically bind toan antigen of interest. During the culture, memory B cells can alsodifferentiate to secrete antibodies (soluble BCR) that bind to thecorresponding antigen-coated microparticles.

Here, the capture ligand present on the microparticles is the antigen ofinterest. Once the BCRs have had an opportunity to bind the antigenpresent on the microparticles, the cells are optionally lysed, and theBCRs are contacted with a detection reagent (in this example, afluorescently-labeled anti-Ig antibody (PE-anti-Ig)).

Any resulting microparticle-antigen-BCR-detection reagent complexes maythen be acquired on a flow cytometer and counted. Any detectedmicroparticle-antigen-BCR-detection reagent complexes indicate thepresence of memory B cells in the co-culture having BCRs thatspecifically bind the antigen of interest. The number and/or proportionof such memory B cells among the starting heterogeneous population ofmemory B cells may be determined based on the number of complexescounted by the flow cytometer. Such an assay may be used to interrogatea cell population of interest for the presence (and optionally, thenumber and/or proportion) of cells having any cell surface protein ofinterest (e.g., any cell surface receptor, where the ligand for thereceptor is present on the microparticles, etc.).

Also provided by the present invention are multiplexed methods usefulfor interrogating a cell population of interest for the presence (andoptionally, the number and/or proportion) of multiple different cellsthat secrete, or present on their surface, various biomarkers ofinterest. One example of a multiplexed method according to an embodimentof the present disclosure is schematically illustrated in FIG. 23. Thisexample involves a heterogeneous population of microparticles, where anintrinsic fluorescent property of each subpopulation of microparticlecorresponds to a specific capture ligand (e.g., antigen, antibody, etc.)disposed on the surface thereof. The intrinsic fluorescent property maybe based, e.g., on the proportion of a first fluorochrome and a secondfluorochrome in the microparticle, as shown on the axes in FIG. 23. Inthis way, a heterogeneous cell population may be interrogated using apanel of capture ligands (e.g., a panel of antigens, such as HLAantigens) to detect cells in the heterogeneous cell population thatsecrete (or present on the surface thereof) a biomarker that binds toany one of the different capture ligands. FIG. 24 illustrates a FlowSpotassay according to one embodiment, while FIG. 23 illustrates how thisapproach may be multiplexed. As shown in FIG. 24, a sample containingmemory B cells may be co-cultured with microparticles having aparticular antigen of interest (here, an HLA antigen of interest)disposed on the surface thereof. BCRs that specifically bind to the HLAantigen are captured by the microparticles via the HLA antigen. Thecomplexes may subsequently be detected and counted in a flow cytometeror a Luminex machine. Such an assay may be multiplexed by co-culturing aheterogeneous population of memory B cells with a heterogeneouspopulation of microparticles that make up a panel of HLA antigens, asshown in FIG. 23.

Utility

The methods of the present disclosure (as well as the compositions,systems and kits described below) find use in a variety of applications,including, e.g., research applications, clinical applications (e.g.,clinical diagnostic applications), etc.

According to certain embodiments, methods of the present disclosure finduse in evaluating (optionally, quantitatively) specific cellular(including immune) response to virus, bacteria, allergens,allo-antigens, auto-antigens, tumors, and the like. In certain aspects,methods of the present disclosure find use in evaluating (optionally,quantitatively) the efficacy of vaccines, a therapy (e.g.,immunotherapy), and the like. According to certain embodiments, methodsof the present disclosure find use in the context of solid organ, bonemarrow, and/or stem cell transplantation. For example, the methodsenable detection, characterization, and/or quantitation of specific,potential or actual, anti-donor/graft immune responses for purposes ofpre- and post-transplant monitoring.

The methods herein have a number of advantages over existing approaches.For example, the co-culture of cells with the microparticles providesdirect cell to ligand contact, so that no dilution effect in solutionoccurs (e.g., dilution of secreted cytokine by diffusion in the medium).The number of specifically responding cells and the amount/concentrationof the biomarker(s) can be quantitated by positively stainedmicroparticles and fluorescence intensity, respectively. The methods donot suffer from steric hindrance due to size or inability to distinguishindividual cells (e.g., cells on top of cells) because the captureligand is present on the much smaller microparticle which flows throughthe cytometer as a single event, one after the other. The need fordedicated (e.g., limited use) and specialized equipment (e.g., anELISPOT reader) or the need to send samples to reference labs (e.g., forELISPOT analysis) is eliminated because flow cytometers are standardequipment and routinely used for enumerating cells/particles withspecific characteristics. Another advantage of the methods of thepresent disclosure is that radioactivity is not required.

Compositions

Aspects of the present disclosure further include compositions. Thecompositions of the present disclosure find a variety of uses, includingin some aspects, practicing the methods of the present disclosure. Assuch, provided are compositions that include any of the cells,microparticles, reagents, etc. described above in relation to thesubject methods, in any desired combination.

According to certain embodiments, provided is a composition thatincludes a culture medium, a population of cells (at least asubpopulation of which expresses or is capable of expressing abiomarker), a cell lysis buffer, and microparticles including a captureligand that specifically binds the biomarker.

The population of cells may be any cell population of interest,including but not limited to immune cells, lymphocytes, T cells, Bcells, natural killer (NK) cells, natural killer T (NKT) cells,macrophages, dendritic cells, monocytes, and platelets.

In certain aspects, the biomarker is a cytokine, an immunoglobulin, ahormone, a growth factor, an enzyme, a protease, a protein, aglycoprotein, a peptide, a CD (cluster differentiation) molecule, areceptor, a nucleic acid, or any combination thereof. When the biomarkeris a cytokine, the cytokine may be an interferon, a chemokine, aninterleukin, a lymphokine, a tumor necrosis factor, or any combinationthereof.

According to certain embodiments, the compositions further include astimulant that stimulates at least a subpopulation of cells to secretethe biomarker. Stimulants that may be present in the subjectcompositions include, but are not limited to, an antigen, a ligand, aprotein, an allergen, a peptide, a lectin, a nucleic acid, a DNA, a RNA,a cell, a sub-cellular component, a microorganism, a drug, a chemicalcompound, an agonist, an antagonist, and any combination thereof.

Any of the compositions of the present disclosure may be present in acontainer (e.g., a tissue culture container). Suitable containersinclude, but are not limited to, tubes, vials, tissue culture plates(e.g., a 96- or other-well tissue culture plates, etc.), petri dishes,etc.

Any of the compositions of the present disclosure may be present in adevice, e.g., an incubator suitable for co-culturing cells andmicroparticles.

Systems

Also provided by the present disclosure are systems. According tocertain embodiments, the systems find use in practicing one or moresteps of the methods of the present disclosure.

In certain aspects, the system (e.g., a flow cytometry system) isadapted to count a number of positive microparticle complexes, where thepositive microparticle complexes include a microparticle, a captureligand, a biomarker, and a fluorescently-labeled detection reagent. Theflow cytometry system is further adapted to determine the total numberof microparticle complexes acquired by the system, calculate thepercentage of positive microparticle complexes among the total number ofmicroparticle complexes, and determine the number and/or proportion ofcells in a cell-microparticle co-culture that included the biomarker. Incertain aspects, the system is further adapted to determine a mean ormedian fluorescence intensity of the positive microparticle complexesacquired by the system, and determine a level of the biomarker presentin the cell-microparticle co-culture.

By “adapted to” is meant that the system includes the components andfunctionality to perform the recited determinations, calculations, etc.For example, in certain aspects, the system includes a processor and acomputer-readable medium (e.g., a non-transitory computer-readablemedium). The computer-readable medium includes instructions executableby the processor to, e.g., count a number of positive microparticlecomplexes, determine the total number of microparticle complexesacquired by the system, calculate the percentage of positivemicroparticle complexes among the total number of microparticlecomplexes, and determine the number and/or proportion of cells in acell-microparticle co-culture that included the biomarker. Thecomputer-readable medium may further include instructions executable bythe processor to determine a mean or median fluorescence intensity ofthe positive microparticle complexes acquired by the system, anddetermine a level of the biomarker present in the cell-microparticleco-culture.

The computer-readable medium (or processor-readable medium) isnon-transitory in the sense that it does not include transitorypropagating signals per se (e.g., a propagating electromagnetic wavecarrying information on a transmission medium such as space or a cable).The media and instructions may be those designed and constructed for thespecific purpose or purposes. Examples of non-transitorycomputer-readable media include, but are not limited to: magneticstorage media such as hard disks, portable flash drives, floppy disks,and magnetic tape; optical storage media such as Compact Disc/DigitalVideo Discs (CD/DVDs), Compact Disc-Read Only Memories (CD-ROMs), andholographic devices; magneto-optical storage media such as opticaldisks; carrier wave signal processing modules; and hardware devices thatare specially configured to store and execute program code, such asApplication-Specific Integrated Circuits (ASICs), Programmable LogicDevices (PLDs), Read-Only Memory (ROM) and Random-Access Memory (RAM)devices.

Kits

As summarized above, the present disclosure provides kits. The kits mayinclude one or more of any of the components/reagents described above inthe section describing the methods of the present disclosure.

According to certain embodiments, the kits include microparticles thatinclude a capture ligand that specifically binds a biomarker ofinterest, and a stimulant capable of stimulating a cell type of interestto secrete the biomarker. The kits may include any additional usefulcomponents and/or reagents, such as a detection reagent thatspecifically binds the biomarker. Such a detection reagent may be, e.g.,an antibody, a ligand, a receptor, or a nucleic acid, and may include adetectable label (e.g., a fluorescent label, a radiolabel, a luminescentagent, or the like). The kit may include a cell lysis buffer to be usedfor removing cells after co-culturing the microparticles with cells.

Components of the kits of the present disclosure may be present inseparate containers, or multiple components may be present in a singlecontainer. For example, the microparticles may be provided in acontainer separate from a container in which the stimulant is provided.The components may be provided in any suitable container(s), such as atube (e.g., vial), in one or more wells of a plate (e.g., a 96-wellplate, a 384-well plate, etc.), or the like.

In addition to the above-mentioned components, a kit of the presentdisclosure may further include instructions for using the components ofthe kit, e.g., to practice the methods of the present disclosure. Forexample, the kit may include instructions for co-culturing themicroparticles and the cells of interest, instructions for stimulatingthe cell type of interest and detecting a complex that includes themicroparticle, the capture ligand, the biomarker, and the detectionreagent, and/or instructions for detecting the complex by flowcytometry. The instructions may be recorded on a suitable recordingmedium. For example, the instructions may be printed on a substrate,such as paper or plastic, etc. As such, the instructions may be presentin the kits as a package insert, in the labeling of the container of thekit or components thereof (i.e., associated with the packaging orsub-packaging) etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g., portable flash drive, DVD, CD-ROM,diskette, etc. In yet other embodiments, the actual instructions are notpresent in the kit, but means for obtaining the instructions from aremote source, e.g. via the internet, are provided. An example of thisembodiment is a kit that includes a web address where the instructionscan be viewed and/or from which the instructions can be downloaded. Aswith the instructions, the means for obtaining the instructions isrecorded on a suitable substrate.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental Materials and Methods

ELISPOT Procedure

Each well was pre-wet with 15 μL of 35% ethanol (v/v in ddH₂O) for onemin, and then rinsed with 150 μL sterile phosphate buffered saline (PBS)three times before the ethanol evaporated. MultiScreen IP plates(Millipore, Cat No. S2EM004M99) were coated with 100 μL (5 μg/mL finalconcentration) anti-IFN-gamma capture antibody in Coating Buffer((1×PBS): 8 g NaCl; 0.2 g KCl; 1.44 g Na₂HPO₄.7H₂O, 0.24 g KH₂PO₄,dissolved in H₂O to a final volume of 1 L. pH 7.2, sterile-filtered (0.2μm-sized pore filter) and stored at 4° C.). Plates were incubatedovernight at 4° C.

Coating Antibody was discarded, and wells washed 1× with 200 μL/wellBlocking Solution (complete tissue culture medium (e.g. RPMI 1640containing 10% Fetal Bovine Serum (FBS) and 1%Penicillin-Streptomycin-L-Glutamine)). 200 μL/well Blocking Solution wasadded and wells were incubated for 2 h at room temperature.

Blocking Solution was discarded. Mitogen or antigen was prepared,diluted in complete tissue culture medium (e.g., RPMI 1640 with FBS,Pen/Strep, and L-glutamine), and 100 μL/well was added to ELISPOT plate.

Cell suspensions were prepared at different densities, (e.g.,1×10⁵-2×10⁶ cells/mL). 100 μL/well of each cell suspension was added toELISPOT plate wells (the cell concentration used was 1×10⁵/mL). ELISPOTplate lid was replaced, and ELISPOT plate was incubated at 37° C., 5%CO₂ and 99% humidity. The duration of the incubation time can be varied(e.g., 2 h-24 h) depending on the nature of the stimulatory cell culturesystem (incubation time presently used was 16 h).

Cell suspension was aspirated, and wells were washed 2× with deionized(DI) water. Wells were allowed to soak for 3-5 min at each wash step.Wells were washed 3× with 200 μL/well Wash Buffer I (1×PBS containing0.05% Tween-20 (0.5 mL Tween-20 per 1 L PBS), and Wash Buffer wasdiscarded. Human IFNγ Detection Antibody (# Cat. 51-1890KZ, BDBiosciences; 2 μg/mL final concentration) was diluted in Dilution Buffer(1×PBS containing 10% FBS), and 100 μL was added per well. Lid wasreplaced and wells were incubated for 2 h at room temperature.

Detection Antibody solution was discarded and wells were washed 3× with200 μL/well Wash Buffer I. Streptavidin-Horseradish Peroxidase (BD™ELISPOT Streptavidin-HRP, Cat. No. 557630) was diluted in DilutionBuffer (1:100), and 100 μL/well diluted Streptavidin-HRP was added. Lidwas replaced and wells incubated for 1 h at room temperature.

Streptavidin-HRP solution was discarded, and wells were washed 4× with200 μL/well Wash Buffer I. Wells were washed 2× with 200 μL/well WashBuffer II (1×PBS). 100 μL of Final Substrate Solution (BD™ ELISPOT AECSubstrate Set Cat. No. 551951) was added to each well. Spot developmentwas monitored from 5-60 min.

No more than 15 min prior to use, one drop (20 μL) of AEC Chromogen wasmixed with each 1 mL of AEC Substrate. Any remaining prepared AECSubstrate was discarded after use. Substrate reaction was stopped bywashing wells with DI water. Plate was air-dried for 2 h—overnight atroom temperature in the dark, until the plate was completely dry. Plateswere stored in the dark prior to analysis. Spots were enumeratedautomatically using an ELISPOT Analyzer.

FlowSpot Procedure Example

1×10⁷-1×10⁸ carboxyl microspheres (Spherotech, CP-35) were transferredto a microcentrifuge tube. Microspheres were pelleted by centrifugationat 20,000×g for 5 min. Supernatant was removed and microspheres wereresuspended in 100 μL dH₂O by vortex and sonication for 30 seconds.Microspheres were pelleted by centrifugation at 20,000×g for 5 min.Supernatant was removed and the microspheres were resuspended in 200 μL100 mM Monobasic Sodium Phosphate, pH 6.2 by vortex. 10 μL of 50 mg/mLSulfo-NHS (Thermo Scientific, Cat No. 24520) diluted in dH₂O was addedto the microspheres and mixed gently by vortex. 10 μL of 50 mg/mL EDC(Thermo Scientific, Cat No. 22980) diluted in dH₂O was added to themicrospheres and mixed gently by vortex. Microspheres were incubated for20 min at room temperature with gentle mixing by vortex at 10 minintervals. Activated microspheres were pelleted by centrifugation at20,000×g for 5 min. Supernatant was removed and the microspheres wereresuspended in 250 μL of 50 mM MES, pH 5.0 (Sigma, Cat No. M2933), byvortex and sonication for approximately 30 seconds. Microspheres werepelleted by centrifugation at 20,000×g for 5 min. Previous wash wasrepeated (for a total of two washes with 50 mM MES, pH 5.0). Supernatantwas removed and the activated and washed microspheres were resuspendedin 200 μL of 50 mM MES, pH 5.0 by vortex and sonication forapproximately 30 seconds.

10-400 μg of the specific capture antibodies (e.g. monoclonal antibodiesagainst interferon gamma (INFγ), interleukin-2 (IL-2), IL-4, IL-5,IL-10, Granzyme B (GrB), etc.) was added to the microspheres and totalvolume was brought up to 500 μl with 50 mM MES, pH 5.0. Couplingreaction was mixed by vortex and incubated at room temperature for 2 hwith constant vortexing on a vortexer. Coupled microspheres werepelleted by centrifugation at 20,000×g for 5 min. Supernatant wasremoved and pelleted microspheres were resuspended in 500 μL of PBS-TBN(PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4) by vortex andsonication for approximately 30 seconds. This was followed by 30 minincubation with constant vortexing at room temperature. Coupledmicrospheres were pelleted by centrifugation at 20,000×g for 5 min.Supernatant was removed, and the microspheres were resuspended in 1 mLof PBS-TBN by vortex and sonication for approximately 30 seconds.Microspheres were pelleted by centrifugation at 20,000×g for 5 min.Previous wash was repeated (for a total of two washes with 1 mLPBS-TBN). Supernatant was removed, and the capture antibody conjugatedmicrospheres were resuspended in 250-1000 μL of PBS-TBN. About1000-10,000 capture microspheres were used for each 0.1×10⁶ cells.

One hundred thousand peripheral blood mononuclear cells (0.1×10⁶ PBMC)or purified lymphocytes were mixed with 7,000 capture microspheres anddistributed into each assay well of a 96-well U bottom plate. The platewas centrifuged at 1,500×g for 5 min and the supernatant was removed.The pellet of cells and capture beads were resuspended in 200 μL culturemedium with or without testing reagent(s) or stimulus(i), then the platewas incubated in a humidified 37° C., 5% CO₂ incubator for 10 min to 7days (usually 2 to 16 h). After incubation, the cells were lysed inNP-40 lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.5%NP-40). The capture beads were washed twice with 3% HBSA (3% BSA inHBSS) and incubated with 50 μL PE-labeled anti-Bio-marker for 60 min atroom temperature. After two washes of 3% HBSA, the beads were acquiredand analyzed on a BD FACSCanto II flow cytometer. The number of positivespots was calculated based on the following formulas:

Percent positive spots (%)=Number of positive particles acquired/Totalnumber of particles acquired×100

Total number of positive spots=Percent positive spots (%)×7,000 (where7,000=total number of particles/test)

Control wells used were as follows:

-   -   a. Negative control-1: beads+media only (no cells)    -   b. Negative control-2: beads+cells+media; without primary        antibody    -   c. Negative control-3: beads+cells+media; control antigen        (non-activator)    -   d. Positive control: cells+media+polyclonal T or B cell        activators (e.g., PHA, PMA)

Example 1: Detection of Phytohaemagglutinin (PHA) Induced InterferonGamma (IFNγ) Spots by ELISPOT and FlowSpot ELISPOT

One hundred thousand PBMC cells from C.T.L. (Cat # CTL-QC1, ShakerHeights, Ohio) were plated into a filter plate well pre-coated with IFNγcapture antibody and cultured for 16 h in the presence of PHA at variousconcentrations. The positive IFNγ spots were enumerated using a C.T.L.ImmunoSpot® Analyzer (FIG. 3 and FIG. 4).

Flowspot

One hundred thousand of the same PBMC cells used in ELISPOT procedurewere co-cultured with 7,000 IFNγ capture microparticles for 16 h in thepresence of PHA at various concentrations. The spots and fluorescenceintensity (FI) of FLOWSPOT were counted and measured by a FACSCanto IIflow cytometer (BD Biosciences). Total number of positive IFNγ spotswere calculated based on the previously described formulas. The FLOWSPOTwas observed to be at least twenty times more sensitive than ELISPOT andable to detect very weak IFNγ responses induced by PHA (FIG. 3 and FIG.4).

Example 2: Peptide Specific IFNγ Spots Detection by FLOWSPOT

One hundred thousand HLA-typed antigen specific T cells from C.T.L. (Cat# CTL-QC1, Shaker Heights, Ohio) were co-cultured with 7,000 IFNγcapture microparticles for 16 h in the presence of the differentpeptides (10 nM) specified in FIG. 5. The IFNγ secretion induced bythree peptides (# Cat. CTL-QC1; C.T.L.) were previously tested againstthe paired PMBCs (C.T.L. cells) by ELISPOT assay in C.T.L. and showedthat HCMV pp65, Flu-Matrix, and influenza A were able to elicit strong,medium, and negative IFNγ responses on the PBMCs respectively. The sameresponse pattern was also observed using FLOWSPOT but yielded atwo-parameter result collected simultaneously in the same reaction well,i.e., the percentage of positive responding cells (% positive spots) andthe relative IFNγ concentration (FI). As shown in FIG. 5, HCMV pp65induced 19% IFNγ positive spots (1,330 spots) with a high Fl (FI=25,286)and Flu-Matrix induced 20% positive spots (1,400 spots) with a weak Fl(FI=678) compared to the negative response induced by influenza A (2%positive spots=140 spots; FI=378) and the media control (1% positivespots=70 spots; FI=443).

Example 3: Two-Color IFNγ/IL-2 Detection by FLOWSPOT

One hundred thousand C.T.L. cells were co-cultured with a mix of IFNγand IL-2 capture microparticles (7,000 each with different fluorescentID codes) for 16 h in the presence of PHA at various concentrations.FIG. 6 shows the dose-dependent responses of IFNγ and IL-2 to polyclonalT cell stimulus PHA.

Example 4: Three-Color IFNγ/IL-2/GrB (Granzyme B) Detection by FLOWSPOT

One hundred thousand PBMC cells were co-cultured with a mix of IFNγ,IL-2, and GrB capture microparticles (7,000 each with differentfluorescent ID codes) for 16 h in the presence or absence of PHA (20ug/mL). The captured IFNγ, IL-2, and GrB on microparticles were detectedby a mix of corresponding PE-labeled antibodies (PE-anti-INF-γ,PE-anti-IL-2, and PE-anti-GrB, BD Bioscience). FIG. 7 shows the numberof positive spots induced by PHA stimulation was 5,320 for IFNγ, 4,340for IL-2, and 630 for GrB respectively. There were no more than 6positive spots observed in the media controls. Results also showed thatthe intensity (amount) of IFNγ secretion was significantly greater thanfor IL-2 with median channel values (MCV) of 653 and 407, respectively.FIG. 5 shows that even when the percentage of positive cells isapproximately the same [e.g., Flu-Matrix 1 (20%) and HCMV pp65 (19%)]the difference in intensity (FI) is significantly different (678 versus25,286, respectively).

Example 5: HLA Antigen Memory B Cell Detection

FIG. 8 depicts the principle of FLOWSPOT for antigen-specific memory Bcell detection. One hundred thousand PBMC cells from donor NT withpositive multiple specificity human leukocyte antigen (HLA) class I andII antibodies (HLA Class I Antibodies: A1, A2, A3, A9, A10, A11, A23,A24, A25, A26, A28, A34, A36, A43, A6602, A68, A69, A80; B8, B12, B14,B15, B16, B18, B21, B22, B35, B39, B40, B41, B45, B46, B50, B54, B55,B56, B60, B61, B62, B64, B65, B70, B71, B72, B75, B76, B78, B82, Bw4;Cw3, Cw9, Cw10; HLA Class II Antibodies: DR4, DR7, DR8, DR9, DR12,DRB3*01:01, DR3*03:01, DR53; DQA1*03; DPA1*02:01) or from donor CC withnegative HLA antibody were co-cultured with single antigen Luminex beadscoated with HLA class-I (147,000 microparticles/test; One Lambda) and-II antigens (139,000/test; One Lambda) for 6 days in a humidified 37°C., 5% CO₂ incubator. After cell lysis and washes, the HLA antibodybound on the HLA antigen beads was detected by adding 100 uL ofPE-labeled Goat-anti human IgG (Jackson ImmunoResearch) and incubated at22° C. for 30 min. The beads were washed and acquired on a FACSCanto IIflow cytometer. As shown in FIG. 9, a total of 13,230 HLA class I and6,975 HLA class II positive spots were detected on NT but none weredetected with negative control CC cells (zero positive spots).

Example 6: Evaluation of Immunotherapeutic Drug Effects on Th1 and Th2Cytokine Secretion by FLOWSPOT

0.1×10⁶ lymphocytes were incubated with a mix of Th1 cytokine (IFN-γ andIL-2) or Th2 cytokine (IL-4, IL-5, and IL-10) capture microparticles(8,000/each with different fluorescent ID codes) in control media or theconditioned media containing various types of immunotherapeutic agents(Solumedrol 100 μM, Pharmacia & Upjohn Co.; Sirolimus 500 ng/mL, Wyethpharmaceuticals Inc.; Prograf 100 ng/mL, Astellas Pharma US, Inc.;Infliximab 1 mg/mL, Janssen Biotech, Inc.) at 37° C. in a 5% CO₂incubator for 16 h with and without adding PHA or P+I (50 ng/mL of PMA+1μg/mL of lonomycin). The cells were lysed with lysis buffer (50 mMTris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40) and washed threetimes with 3% HBSA wash buffer (3% Bovine Serum Albumin in Hank'sBalanced Salt Solution), the capture microparticles were then incubatedwith 50 μL detection antibodies mix (PE-anti-INF-γ, PE-anti-IL-2,PE-anti-IL-4, PE-anti-IL-5 and PE-anti-IL-10) from BD Bioscience at RTfor 1 h with shaking. After three more washes, the microparticles wereacquired and analyzed on a BD FACSCanto II flow cytometer. The resultwas expressed as a percentage of each of the different positive cytokinecapture microparticles. FIG. 10-FIG. 14 depict the differential effectsof the immunotherapeutic agents on the secretion of Th1 and Th2cytokines.

Example 7: Evaluation of Specific Immunity to Virus Vaccine, Bacterial,and Tumor Antigens by FLOWSPOT

0.1×10⁶ PBMC from each of three donors (CC, SH, and FM) were incubatedwith a mix of Th1 cytokine (IFN-γ and IL-2) or Th2 cytokine (IL-4, IL-5,and IL-10) capture microparticles (8000/each with different fluorescentID codes) in control media or the conditioned media containing varioustypes of antigens (45 μg/mL Fluzone, Sanofi Pasteur Inc.; 25 μg/mL ofMycobacterium tuberculosis Ag 6 KDa, ADJ; 25 μg/mL of Mycobacteriumtuberculosis Ag 16 KDa, ADJ; 25 μg/mL of Tetanus Toxoid Protein, ADJ; 25μg/mL of Diphtheria Toxoid Protein, ADJ; and 6.25 μg/mL of Testis CancerAntigen, NY-ESO-1, JPT) at 37° C. in a 5% CO₂ incubator for 16 h. Thecells were lysed with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl,2 mM EDTA, 1% NP-40) and washed three times with wash buffer, thecapture microparticles were then incubated with 50 μL mix of Th1detection antibodies (PE-anti-INF-γ and PE-anti-IL-2) or Th2 detectionantibody mix (PE-anti-IL-4, PE-anti-IL-5 and PE-anti-IL-10) at RT for 1h with shaking. After three more washes, the microparticles wereacquired and analyzed on a BD FACSCanto II flow cytometer. The finalresult (FIG. 15-FIG. 19) was expressed as percentage of positivemicroparticles compared to the total number of microparticle complexes.Results demonstrated that the specific immunities to various antigens ofvirus, bacteria, and tumor are significantly different in three donors.

Example 8: Detection of Virus Peptide/Antigen Specific T Cells Responseby FLOWSPOT

0.1×10⁶ PBMC from each of 7 blood donors were incubated with a mix ofTh1 cytokine (IFN-γ and IL-2) or Th2 cytokine (IL-4, IL-5, and IL-10)capture microparticles (8000/each with different fluorescent ID codes)in control media or the conditioned media containing various types ofvirus peptides and antigen purchased from C.T.L. (5 nM Influenza A PApeptide, 500 nM HCMV-pp65, 30 ug/mL HCMV-Gr2 Ag) at 37° C. in a 5% CO₂incubator for 16 h. The cells were lysed with lysis buffer (50 mMTris-HCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40) and washed three timeswith wash buffer, the capture microparticles were then incubated with 50μL mix of Th1 detection antibodies (PE-anti-INF-γ and PE-anti-IL-2) orTh2 detection antibody mix (PE-anti-IL-4, PE-anti-IL-5 andPE-anti-IL-10) at RT for 1 h with shaking. After three more washes, themicroparticles were acquired and analyzed on a BD FACSCanto II flowcytometer. The results (FIG. 5, FIG. 20-FIG. 22) show that the responsepatterns of Th1 and Th2 cytokine secretion varies in different donorsagainst different virus peptides and antigens.

Example 9: Multiplex HLA Antigen Specific Memory B Cell Detection

Principle of Assay

FIG. 23 and FIG. 24 depict the principle of the assay, as describedbelow. The biological sample containing memory B cells was incubatedwith a mix of uniquely labeled fluorescent beads, each conjugated withdifferent purified HLA antigens. The HLA antigen specific memory B cellscan be captured by the corresponding HLA antigen beads through theinteraction of membrane B cell receptors (mBCR) and HLA antigens on thebead surface(s). After cell lysis, the captured mBCR on each HLA antigenbead can be detected by fluorescent anti-immunoglobulin in a flowcytometer. The number and fluorescence intensity of positive beads areproportional to the number of HLA antigen specific memory B cells.

Methods and Results

One hundred thousand PBMC cells from donors positive (NT) (FIG. 26) ornegative (RT) (FIG. 25) for multiple HLA class I antibody specificities(HLA Class I Antibodies: A1, A2, A3, A9, A10, A11, A23, A24, A25, A26,A28, A32, A34, A36, A43,

A6602, A68, A69, A80; B8, B12, B14, B15, B16, B18, B21, B22, B35, B39,B40, B41, B45, B46, B50, B54, B55, B56, B60, B61, B62, B64, B65, B70,B71, B72, B75, B76, B78, B82, Bw4; Cw3, Cw9, Cw10) were co-culturedindependently and concurrently with 97 single HLA class-I antigenscoated Luminex beads (147,000 beads/test; One Lambda) for 16 h in ahumidified 37° C., 5% CO₂ incubator. After cell lysis and washes, theHLA antibodies secreted from plasma cells and/or IgG B cell receptors(BCR) bound on each single HLA antigen beads were labeled by adding 100uL of PE-labeled Goat-anti human IgG (Jackson ImmunoResearch) andincubated at 22° C. for 30 min. The final washed beads were acquired ona FACSCanto II flow cytometer. The (97) bead populations were gated on adot plot and the percentage of positive FLOWSPOTS on each single HLAantigen bead population was calculated by using Diva software (BDBiosciences).

A total of 27 HLA-A (FIG. 27) and 35 HLA-B (FIG. 28) specific beadpopulations were detected positive by FLOWSPOT in NT cells but negativein RT cells. The positive FLOWSPOT populations were concordant withserum HLA antibody screening results by Luminex single antigen beadsassay.

Example 10: INF-y Secretion Detection by CBA and FlowSpot

Cytometric Bead Array (CBA): INF-y Secretion Detection in Culture Media

One hundred thousand peripheral blood mononuclear cells (0.1×10⁶ PBMC)in 100 μL of 10% FBS RPMI 1640 containing varying concentrations of PHAwere seeded in each well in a 96 well plate and incubated in ahumidified 37° C., 5% CO₂ incubator for 16 h. The plate was centrifugedat 2,500 g for 3 min and 50 μL culture medium of each well was carefullytransferred into each corresponding test well in a new 96 well plate.After adding 50 μL INFγ capture beads (BD Biosciences; Cat. 558269) intoeach well, the plate was incubated at room temperature (RT) for 60 minwith constant shaking on a plate shaker. After adding 50 μL INFγPE-detection reagents (BD Biosciences; Cat. 51-9004031), the plate wascontinually incubated at RT for additional 2 h. The beads were thenwashed three times with 200 μL wash buffer (0.05% Tween-20 in PBS) andacquired on a BD FACSCanto II flow cytometer. The concentrations of INFγin the culture media from each well were extrapolated from the standardcurve generated from a series of known INFγ standards in the same batchof CBA assay. Results are shown in FIG. 29.

FlowSpot: INF-γ Secretion In Situ Detection

In parallel, 0.1×10⁶ PBMC cells from the same donor were co-culturedwith 7,000 IFNγ capture microparticles in a humidified 37° C., 5% CO₂incubator for 16 h in the presence of PHA at various concentrations.After incubation, the cells were lysed in NP-40 lysis buffer (10 mMTris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.5% NP-40). The capture beadswere washed twice with 3% HBSA (3% BSA in HBSS) and incubated with 50 μLPE-labeled anti-IFNγ for 60 min at room temperature. After an additionaltwo washes of 3% HBSA, the beads were acquired and analyzed on a BDFACSCanto II flow cytometer. The number of positive spots is calculatedbased on the previously described formulas, and results are shown inFIG. 29.

A comparison of IFNγ Detection by FLOWSPOT and CBA is shown in Table 1below.

TABLE 1 Comparison of IFN_(γ) Detection by FLOWSPOT and CBA Comparisonof IFN_(γ) Detection by FLOWSPOT and CBA FLOWSPOT CBA PHA % PositiveSPOTS IFN_(γ) (μg/mL) Spot (n) MFI (pg/ml) 0 0 0 0 10 0.5 24 1680 166 171 35 2450 659 36 2.5 43 3010 4050 76 5 45 3150 9541 174 10 56 3920 19594281 15 57 3990 21680 344 20 55 3850 22241 312

The BD™ Cytometric Bead Array (CBA) is a multiplex detection system toquantify multiple proteins in a biological sample simultaneously. CBA isa uniform (homogeneous) testing system in which the analytes are evenlydistributed in a liquid sample and all capture microparticles have asame probability to capture the analytes. In contrast to CBA, Flowspotis a non-uniform (or inhomogeneous) detection system. Depending on thedistribution of microparticles and cells, the capture microparticlesadjacent to the biomarker secreting cells (e.g., responding cells) havea higher probability, due to proximity, to capture the biomarkerssecreted from the responding cells than the cells that are distant fromthe responding cells, as well as a higher probability to saturate moreof the capture ligands due to high proximal biomarker concentration.Therefore, the percentage of positive reacting microparticles isproportionately correlated to the number of reacting cells; and thefluorescence intensity of positive microparticles reflects the relativebiomarker secreting capability of the responding cells. Compared to CBA,Flowspot is more sensitive and more specific.

Example 11: Evaluation of Specific Immunity to Donor Cells by Flowspot

Recipient (R) Cells:

HLA Type: A*02:01, 03:01; B*07:02, 37:01; Bw4, w6; C*06:02, 07:02;DRB1*15:01, X; DR51; DQB1*06:02, X; DPB1*02:01, 04:01; DPA1*01:03/10, X

Donor (D) Cells:

HLA Type: A*26:01, 30:01; B*13:02, 38:01; Bw4; C*06:02, 12:03;DRB1*04:03, 13:01; DR52, 53; DQB1*03:05, 06:03; DPB1*02:01, 04:01

Procedure:

0.1×10⁶ of recipient, R, PBMCs were co-cultured with an equal number of50 Gy gamma irradiated autologous (iR) or donor (iD) PBMCs and a mix ofTh1 cytokine (IFN-γ and IL-2) capture microparticles (8,000/each withdifferent fluorescent ID codes) at 37° C. in a 5% CO₂ incubator for 72h. The cells were lysed with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mMNaCl, 2 mM EDTA, 1% NP-40) and washed three times with 3% HBSA washbuffer (3% Bovine Serum Albumin in Hank's Balanced Salt Solution). Thecapture microparticles were then incubated with 50 μL detectionantibodies mix (PE-anti-INF-γ and PE-anti-IL-2) from BD Bioscience at RTfor 1 h with shaking. After three more washes, the microparticles wereacquired and analyzed on a BD FACSCanto II flow cytometer. The resultswere expressed as the percent positive of the total and the medianfluorescent intensity (MFI) for each of the different cytokine capturemicroparticles.

As shown in FIG. 30, the recipient has a negative response to irradiatedautologous cells (R+iR) but a strong positive response to irradiateddonor cells (R+iD) for both interferon gamma (IFN-γ) and interleukin-2(IL-2) secretion.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1.-28. (canceled)
 29. A method, comprising: co-culturing in a culturemedium: a heterogeneous cell population comprising a first subpopulationof cells that express a biomarker and a second subpopulation of cells;and microparticles comprising capture ligands that specifically bind tothe biomarker.
 30. The method according to claim 29, wherein the firstsubpopulation of cells secrete the biomarker.
 31. The method accordingto claim 30, further comprising stimulating the first subpopulation ofcells to secrete the biomarker, wherein upon secretion of the biomarker,the biomarker is bound by the capture ligands.
 32. The method accordingto claim 31, wherein the stimulating comprises adding a stimulant to theculture medium.
 33. The method according to claim 32, wherein thestimulant is selected from the group consisting of: an antibody, anantigen, a ligand, a protein, a lectin, a nucleic acid, a drug, anallergen, a peptide, an interferon, a chemokine, an interleukin, alymphokine, a tumor necrosis factor, a CD molecule, a cell, a vaccine, aparasite, a fungus, a sub-cellular component, a virus, a microorganism,a chemical compound, an agonist, an antagonist, and combinationsthereof.
 34. The method according to claim 29, wherein the biomarker ispresented on the surface of the first subpopulation of cells, whereinupon presentation of the biomarker on the surface of the firstsubpopulation of cells, the biomarker is bound by the capture ligands.35. The method according to claim 29, further comprising, after thebiomarker is bound by the capture ligands, lysing the cells of theheterogeneous cell population.
 36. The method according to claim 29,further comprising, after the biomarker is bound by the capture ligands,contacting the biomarker with a detection reagent to form complexescomprising the microparticle, the capture ligand, the biomarker, and thedetection reagent.
 37. The method according to claim 36, furthercomprising detecting the complexes.
 38. The method according to claim37, wherein the detecting is by flow cytometry or by Luminex machine.39. The method according to claim 37, wherein the detecting is by masscytometry.
 40. The method according to claim 37, wherein the detectingcomprises counting the number of positive complexes.
 41. The methodaccording to claim 40, comprising determining the proportion of thefirst subpopulation of positive cells among the heterogeneous cellpopulation based on the number of complexes counted.
 42. The methodaccording to claim 37, comprising determining a signal intensity of eachdetected complex.
 43. The method according to claim 42, comprisingdetermining a level of the biomarker secreted by the first population ofcells based on the mean or median signal intensity of the detectedcomplexes.
 44. The method according to claim 29, wherein the biomarkeris selected from the group consisting of: a cytokine, an immunoglobulin,a hormone, a growth factor, an enzyme, a protease, an allergen, aprotein, a nucleic acid, tumor marker, a CD molecule, and a combinationthereof.
 45. The method according to claim 44, wherein the biomarker isa cytokine selected from the group consisting of: an interferon, achemokine, an interleukin, a lymphokine, a tumor necrosis factor, aperforin, a granzyme, and combinations thereof.
 46. The method accordingto claim 29, wherein the cells of the first subpopulation of cells areof a type selected from: immune cells, lymphocytes, T cells, B cells,natural killer (NK) cells, natural killer T (NKT) cells, macrophages,dendritic cells, monocytes, and platelets.
 47. The method according toclaim 29, wherein the microparticles have a greatest dimension of from0.001 μm to 20 μm.
 48. The method according to claim 29, wherein thecapture ligands are selected from the group consisting of: antibodies,antigens, enzymes, substrates, allergens, peptides, nucleic acids,drugs, chemical compounds, and combinations thereof. 49.-68. (canceled)